Synthesis of functionalizable or functionalized poly(3,4-ethylenedioxythiphene)-based polymers and monomers therefor

ABSTRACT

A method of forming a compound having the formula:includes the reaction:n the presence of a base comprising teat-butyl lithium, lithium diisopropylamide, sodium hydroxide, potassium hydroxide, lithium hydroxide, a potassium alkoxide or a sodium alkoxide to achieve a yield of at least 90%, wherein X is a halo atom selected from the group consisting of Cl, Br and I.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 16/315,954, filed Jan. 7, 2019, which is a nationalphase filing of PCT International Patent Application No.PCT/US2017/041286, filed Jul. 10, 2017, which claims benefit of U.S.Provisional Patent Application Ser. No. 62/359,866, filed Jul. 8, 2016,the disclosures of which are incorporated herein by reference.

GOVERNMENTAL INTEREST

This invention was made with government support under grant no. NS062019awarded by the National Institutes of Health. The government has certainrights in this invention.

BACKGROUND

The following information is provided to assist the reader inunderstanding technologies disclosed below and the environment in whichsuch technologies may typically be used. The terms used herein are notintended to be limited to any particular narrow interpretation unlessclearly stated otherwise in this document. References set forth hereinmay facilitate understanding of the technologies or the backgroundthereof The disclosure of all references cited herein are incorporatedby reference.

Since the invention at Bayer AG in late 1980s, the conducting polymerpoly 3,4-ethylenedioxythiophene (PEDOT) and its derivatives have formedone of most popular categories of commercially available conductingpolymeric materials for decades. Numerous applications have beenachieved in a broad ranue of fields, from organic field-effecttransistors, solar cells and light emitting diodes to nanomedicine,biosensors and bioelectronics.

In the pursuit of high efficiency and functionality of PEDOT baseddevices, especially in the research frontiers of biomedical field, it ishighly desired to have a conducting material with readily availablereactive groups for further modification and bioconjugations. However,PEDOT lacks reactive sites for direct functionalization, while thesupply of commercially available 3,4-ethylenedioxythiophene (EDOT)derivatives, such as hydroxymethyl EDOT (EDOT-OH) and EDOT-acid, arelimited and costly (about $300/g). The high cost has been the result ofknown synthesis strategies that often involve multiple reaction stepsand low yield. Such challenges prevent bulk production and limit theavailability and potential for widespread applications. It is verydesirable to develop more convenient and cost-effective synthetic routesfor large scale production of functionalized PEDOT derivatives.

SUMMARY

In one aspect, a method of forming the compound having the formula:

(sometimes referred to herein as exomethylene functionalized3,4-ethylenedioxythiophene, exomethylene functionalized EDOT, orEDOT-EM) includes the reaction:

in the presence of a base, wherein X is a halo atom selected from thegroup consisting of Cl, Br and I. In that regard, a2-halomethyl-2,3-dihydrothieno[3,4-b][1,4]dioxine compound reacts in thepresence of a base to form EDOT-EM. In a number of embodiments, X is Cl.In a number of embodiments, the conjugate acid of the base has a pKa ofat least 17. Such pKa information is readily available in published pKatables or readily determinable using well-known procedures. The reactionmay, for example, take place under conditions to limit nucleophilicaddition reactions by the base. In a number of embodiments, the base isa non-nucleophilic base. In general, a non-nucleophilic base is a basewhich is a poor nucleophile.

In a number of embodiments, the base is selected from the group ofpotassium tert-butoxide, sodium tert-butoxide, teat-butyl lithium,lithium diisopropylamide, sodium hydroxide, potassium hydroxide, anyother potassium alkoxide or any other sodium alkoxide. In a number ofembodiments, the base is, for example, selected from the group ofpotassium tert-butoxide, sodium tert-butoxide, another potassiumalkoxide or another sodium alkoxide

The reaction may, for example, occur at a temperature in the range ofapproximately 0° C. to 100° C. In a number of embodiments, the reactiontakes place at room temperature. In a number of embodiments, thereaction has a yield of at least 50%, at least 70%, at least 80%, atleast 90%, or at least 95%.

In another aspect, a method of forming a polymer, comprising:polymerizing monomers including at least one of exomethylenefunctionalized 3,4-ethylenedioxythiophene or the reaction product ofexomethylene functionalized 3,4-ethylenedioxythiophene and a compoundincluding a uroup reactive with the exomethylene group of exomethylenefunctionalized 3,4-ethylenedioxythiophene via a polymerization reaction.Such monomers may be represented by the following formulas (wherein theR is the reaction product of the exomethylene group and the compoundincluding a group reactive with the exomethylene group):

In the case that compound G is polymerized, compound G may be foriiiedby reacting compound F with a functionalizing compound including a groupreactive with an exomethylene group of compound F. In a number ofembodiments, the monomers have a purity of at least 90% or at least 95%in the polymerization reaction. In a number of embodiments, theconcentration of the monomers in the polymerization reaction is at least20 mM, 30 mM, 40 mM, 50 mM or 60 mM. In a number of embodiments,electrolyte concentration is in the range of 10 mM to 1 M, or in therange of 50 mM to 200 mM. In a number of embodiments, an electrolyteconcentration in the polymerization is at least 100 mM. Suitable typesof electrolytes include, but are not limited to lithium, sodium,potassium, tetrabutylammonium or tetrabutylphosphonium salts with avariety of counter ions, including, for example, perchlorate, nitrate,acetate, chloride, bromide, iodide, hexafluorophosphate,tetrafluoroborate, tetraphenylborate, or polystyrene sulfonate (PSS).Other suitable electrolytes include ionic liquids such as ammonium-,pyridinium-, imidazolium-, or phosphonium-containing ionic liquids. Theelectrolyte may, for example, be LiClO₄.

Solvent systems/solvent mixtures for use in the polymerization reactionshereof include, for example mixtures of water and acetonitrile, ethanol,dimethylformamide, propylene carbonate, etc. Binary solvents (that is,mixtures of organic solvent and water) may, for example, include 5% to95% of water. In a number of embodiments, a solvent system/solventmixture of acetonitrile and water may be used. The acetonitrile andwater may, for example, be present in a 1:1 mixture.

A potentiostatic polymerization method was used in a number ofembodiments. A potential for such a reaction may, for example, be in therange of 0.6 V to 1.5 V, or in the range of 0.9 V to 1.2 V. In a numberof potentiostatic polymerizations hereof, the potential may, forexample, be approximately 1.1V. Electropolymerization methods such aspotentiostatic or cyclic voltammetry (for example, at the same potentialrange as set forth above) may be used to synthesize the polymers hereofFurther, galvanostatic polymerization methods may be used with, forexample, a current density in the range of 1 μA/cm² to 100 μA/cm². In anumber of embodiments hereof, galvanostatic polymerization may occur ata current density of 50 μA/cm².

In a number of embodiments, the monomers are polymerized viaelectropolymerization. The method may, for example, further includefunctionalization of the polymer after synthesis by reacting a compoundwith an exomethylene group of the polymer in the case that at least someof the monomers are not functionalized prior to polymerization.

In another aspect, a method of forming a functionalized polymer includesreacting a polymer comprising the repeat group:

with at least one compound having a group reactive with an exomethylenegroup of the polymer. The polymer may, for example, be reacted with theat least one compound in a solution state. In a number of embodiments,the group reactive with the exomethylene group is a thiol group. The atleast one compound may, for example, be a polymer. The polymer hereof(PEDOT-EM polymers) may, for example, be post-functionalized withvarious thiol-containing small molecules. Examples of readily availablesmall thiol compounds include, but are not limited, to3-mercaptopropionic acid, 3-chloro-1-propanethiol,1-mercapto-2-propanol, 3-mercapto-1-propanol, 3-amino-1-propanethiolhydrochloride, 4-mercapto-1-butano, 6-mercaptohexanoic acid,6-mercapto-1-hexanol, 6-mercapto-1-hexanol, 6-amino-1-hexanethiolhydrochloride, 8-mercaptooctanoic acid, 8-mercapto-1-octanol,8-amino-1-octanethiol hydrochloride, 9-mercapto-1-nonanol,11-mercaptoundecanoic acid, 11-mercaptoundecanamide,11-azido-1-undecanethiol, 11-mercapto-1-undecanol,11-amino-1-undecanethiol hydrochloride, 11-mercaptoundecylphosphonicacid, 11-mercaptoundecylphosphoric acid, 12-mercaptododecanoic acid,1-(11-mercaptowidecypimidazole,(11-mercaptoundecyl)-N,N,N-trimethylammonium bromide,11-(1H-pyrrol-1-yl)undec ane-1-thiol, 6-(ferrocenyl)hexanethiol, 12-mereaptododecanoic acid NHS ester, 16-mercaptohexadecanoic acid,16-mercaptohexadecanamide, 16-amino-1-hexadecanethiol hydrochloride,11-mercaptoundecylhydroquinone, triethylene glycolmono-11-mercaptoundecyl ether, (11-mercaptoundecyptetra (ethyleneglycol), 11-(ferrocenyl)undecanethiol, (11-mercaptoundecyl)hexa(ethyleneglycol). The functionalized polymers may, for example, be reacted withzwitterionic amino acid cysteine for non-specific biofouling resistanceof the surface. Likewise, the polymers may be reacted with variousbiomolecules (for example, proteins, nucleic acids etc). The polymermay, for example, be reacted with a biomolecule such as an aptamer foruse, for example, in real time neurotransmitter detection, or with oneor more peptides for use, for example, in improving biocompatibility ofthe coated substrate with enhanced neuronal survival. Once again, theexomethylene group of the monomer may be reacted with suchfimctionalizing compound or other fiinctionalizinu compounds reactivewith the exomethylene group to create the substituent R set forth abovein compound B.

The present systems, methods and compositions, along with the attributesand attendant advantages thereof, will best be appreciated andunderstood in view of the following detailed description taken inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates synthesis of EDOT-EM in a previously published methodproceeding through compounds B to E to EDOT-EM, wherein, in thesynthetic method hereof, EDOT-EM is synthesized directly fromrepresentative compound B (as highlighted in the boxed scheme) with manyfewer steps, faster reaction rate, quantitative conversion and extremelyhigh yield.

FIG. 2 illustrates a polymerization curve of EDOT-EM through agalvanostatic method.

FIG. 3A illustrates a comparison of electrochemical impedance spectra(Bode plots) for PEDOT, wherein data for bare gold substrate are shownin squares and data for polymer-coated gold substrate are shown incircles.

FIG. 3B illustrates a comparison of electrochemical impedance spectra(Bode plots) for PEDOT-EM samples, wherein data for bare gold substrateare shown in squares and data for polymer-coated gold substrate areshown in circles.

FIG. 4 illustrates FTIR spectra of PEDOT film, PEDOT-EM film, andPEDOT-EM film after functionalization with mercaptopropionic acid (MPA),wherein all films were obtained from electrochemical polymerization onAu—Si.

FIG. 5A illustrates a representative optical microscope image of primaryneuron growth on NC-Laminin after two days in culture.

FIG. 5B illustrates a representative optical microscope image of primaryneuron growth on PEDOT-EM-Laminin after two days in culture.

FIG. 5C illustrates a representative optical microscope image of primaryneuron growth on PEDOT-Laminin after two days in culture.

FIG. 6 illustrates photomicrographs providing a comparison of neuroncell attachment and growth between laminin-functionalized PEDOT-EM(left) and unfunctionalized or bare PEDOT-EM (right).

FIG. 7 illustrates photomicrograph demonstrating high density neuroncell attachment via blue DAPI stain (right photomicrograph) andsignificant neurite outgrowth via green beta tubulin stain (leftphotomicrograph) for PEDOT-EM functionalized with L1 protein viacrosslinkers.

FIG. 8 illustrates a comparison of square wave voltammetry (SWV) beforeand after post-functionalization of PEDOT-EM with DNA aptamer based onmethylene blue oxidation.

DESCRIPTION

The present systems, methods and compositions, along with the attributesand attendant advantages thereof, will best be appreciated andunderstood in view of the following description taken in conjunctionwith any accompanying drawings.

In a number of embodiments, methods hereof provide for the synthesis ofEDOT monomer having an exomethylene functional group (EDOT-EM),polymerization of such a monomer (or a functionalized derivative of sucha monomer) and post-polymerization functionalization of polymersproduced from such monomers.

It will be readily understood that the components of the embodiments, asgenerally described and illustrated in the figures herein, may bearranged and designed in a wide variety of different configurations inaddition to the described example embodiments. Thus, the following moredetailed description of the example embodiments, as represented in thefigures, is not intended to limit the scope of the embodiments, asclaimed, but is merely representative of example embodiments.

Reference throughout this specification to “one embodiment” or “anembodiment” (or the like) means that a particular feature, structure, orcharacteristic described in connection with the embodiment is includedin at least one embodiment. Thus, the appearance of the phrases “in oneembodiment” or “in an embodiment” or the like in various placesthroughout this specification are not necessarily all referring to thesame embodiment.

Furthermore, described features, structures, or characteristics may becombined in any suitable manner in one or more embodiments. In thefollowing description, numerous specific details are provided to give athorough understanding of embodiments. One skilled in the relevant artwill recognize, however, that the various embodiments can be practicedwithout one or more of the specific details, or with other methods,components, materials, et cetera. In other instances, well knownstructures, materials, or operations are not shown or described indetail to avoid obfuscation.

As used herein and in the appended claims, the singular forms “a,” “an”,and “the” include plural references unless the context clearly dictatesotherwise. Thus, for example, reference to “a compound” includes aplurality of such compounds and equivalents thereof known to thoseskilled in the art, and so forth, and reference to “the compound” is areference to one or more such compounds and equivalents thereof known tothose skilled in the art, and so forth. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range. Unlessotherwise indicated herein, and each separate value as well asintermediate ranges are incorporated into the specification as if itwere individually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contraindicated by the text.

The inventors hereof have discovered a simplified synthetic route formaking EDOT-EM, which is a highly versatile monomer through which a widevariety of EDOT and PEDOT derivatives may be synthesized. A previouslyreported method for synthesizing EDOT-EM required five steps with lowyields in these steps, while the present synthetic method may proceed inonly a single step with a yield of ˜100% at room temperature and largequantity. A comparison of the novel synthetic approach hereof and apreviously reported method is illustrated in FIG. 1.

The inventors have also shown that the EDOT-EM monomer can be easilyfunctionalized. The targeting compound EDOT-EM is a versatileintermediate that can be facilely fimctionalized on demand through, forexample, thiol-ene click chemistry, from small molecules tomacromolecular proteins. In a number of embodiments, any molecule in the“tool-box” equipped with free thiol (SH) units may be attached, via, forexample, click chemistry. The monomer itself may, for example, beelectropolymerized into conductive polymer and be post-functionalizedwith desired biomolecules such as peptides and proteins.

EDOT-OH is one of the earliest developed and most widely used EDOTintermediates. EDOT-OH was first synthesized from cyclization of diethyl3,4-dihydroxythiophene-2,5-dicarboxylate through either a Williamsonether synthesis or Mitsunobu reaction pathway, followed bydecarboxylation. However, the overall yield was relatively low.Subsequently, an alternative method was developed to synthesize EDOT-OHfrom 3,4-dimethoxythiophene as the starting material, through an acidcatalyzed transesterification pathway as show in scheme proceedingthrough substituents A-E of FIG. 1. See, for example, Lu, Y.; Wen,Y.-p.; Lu, B.-y.; Duan, X.-m.; Xu, J.-k.; Zhang, L.; Huang, Y. Chin JPolyin Sci 2012, 30, 824.

Beverina and co-workers previously carried out the synthesis of EDOT-EMwith the commercially available EDOT-OH as the starting material. Sassi,M.; Mascheroni, L.; Ruffo, R.; Salamone, M. M.; Pagani, G. A.; Mari, C.M.; D'Orazio, G.; La Feria, B.; Beverina, L. Organic Letters 2013, 15,3502. As discussed above, this starting material is very expensive(possibly as a result of the 3 steps of synthesis needed from rawmaterial and the low yield). After a tosylation of hydroxyl group onEDOT-OH followed by elimination of tosylate group, EDOT-EM was obtainedwith a two-step yield around 52% (see the synthetic scheme of FIG. 1).Thus, compared with the published method for the production of EDOT-EM,the present methodology requires fewer reactions, with faster reactionrate, quantitative conversion and extremely high yield. The presence ofEM group in EDOT-EM provides many possibilities for derivatization by,for example, either hydro-alkoxy addition or thiol-ene click chemistry.

In the process of synthesizing EDOT-OH (compound D), during a reactionof 2-chloromethyl-2,3-dihydrothieno[3,4-b][1,4]dioxine (EDOT-MeCl,compound B) with sodium acetate to make compound C (see FIG. 1), at 120°C. in DMSO, the conversion rate to targeting compound C was lower thanexpected, although the reactant was completely consumed. Surprisingly, a“side product” showed even lower polarity than the reactant EDOT-MeCl(compound B). After isolation and purification, the obtained sideproduct showed same number of carbons but one less proton compared toEDOT-MeCl, based on ¹H and ¹³C nuclear magnetic resonance (NMR) spectra.From Distortionless Enhancement by Polarization Transfer (DEPT) 135 NMRspectrum, two CH₂ carbons were found. It was suspected that the compoundcould be a product from dehydrohalogenation reaction upon heating athigh temperature. Soon after, detailed atomic connectivity was obtainedfrom two-dimensional NMR heteronuclear single quantum coherence orheteronuclear single quantum correlation (HSQC) studies andheteronuclear multiple bond correlation (HMBC) studies, which confirmedthat hypothesis. Without limitation to any mechanism, it is proposedthat, after elimination of HCl, the product converted into anEDOT-alkene derivative EDOT-EM, and the newly-formed double bond couldextend the conjugation and thus stabilize the overall molecularstructure.

As shown in table 1, three different types of reagents were tested tostudy optimization of the reaction conditions to produce EDOT-EM at highyield. First, a non-nucleophilic strong base potassium tert-butoxide(tBuOK) was applied. Dehydrohalogenation was completed within 30 minutesat room temperature with above 95% isolation yield after washing andpurification with silica gel chromatography. Second, reactions wereattempted with milder bases (either sodium hydroxide or potassiumhydroxide). Conversion rate was slow at room temperature but could becompleted after heating at 90° C. in a sealed reaction vessel with ayield of 92%. Without limitation to any mechanism, the lower yield maybe a result of a small amount of oligomerization or polymerization ofEDOT-EIVI during heating. Finally, a weak non-nucleophilic base,diisopropylamine, was tested at both room temperature and heatedcondition. Reaction conversion was low and the product was not isolated.The results indicated that strong non-nucleophilic bases (pKa ofconjugate acid around 17, such as sodium hydride sodium tert-butoxideand potassium tert-butoxide) are desirable for production of EDOT-EM athigh yield in the present reaction schemes. Reagents with greaterbasicity (pKA of conjugate acid around 35-40, such as tert-butyl lithiumand lithium diisopropylamide) were not tested. It is believed that inthe presence of stronger bases the reaction will occur in the samemanner but may be carried out at lower temperature. With moderate,non-nucleophilic bases (pKa of conjugate acid in the range ofapproximately 10-13 and below), reactions are slower than with strongerbases. Although sodium hydroxide and potassium hydroxide are strongnucleophiles, E2-type eliminations still proceed very well under bothconditions with elevated temperature in the sealed reaction vessel.

TABLE 1 Reagent Reaction Time Solvent Temperature Yield tBuOK 30 min DryTHF RT >95% NaOH/KOH Overnight Methanol 90° C. (sealed)  92%Diisopropylamine Overnight Methanol 90° C. (sealed) —

A generalized scheme hereof for synthesis of EDOT-EM (compound F) is setforth below as

As described above, the reaction of compound B′(2-halomethyl-2,3-dihydrothieno[3,4-b][1,4]dioxine) occurs in thepresence of a base and X is a halo atom selected from the groupconsisting of Cl, Br and I. In a number of embodiments (as, for example,described in connection with FIG. 1), X is Cl. In a number ofembodiments, the conjugate acid of the base has a pKa of at least 17.Such pKa information is readily available in published pKa tables orreadily determinable using well-known procedures. The reaction may, forexample, take place under conditions to limit nucleophilic additionreactions by the base. In a number of embodiments, the base is anon-nucleophilic base. In general, a non-nucleophilic base is a basewhich is a poor nucleophile.

In a number of embodiments, the base is selected from the group ofpotassium tert-butoxide, sodium tert-butoxide, tert-butyl lithium,lithium diisopropylamide, sodium hydroxide, potassium hydroxide, anyother potassium alkoxide or any other sodium alkoxide. The base may, forexample, be selected from the group of potassium tert-butoxide, sodiumtert-butoxide, another potassium alkoxide or another sodium alkoxide.The reaction may, for example, occur at a temperature in the range ofapproximately 0° C. to 100° C. In a number of embodiments, the reactiontakes place at room temperature. In a number of embodiments, thereaction has a yield of at least 50%, at least 70%, at least 80%, atleast 90%, or at least 95%.

Electro-polymerization methods were employed for their simplicity andexcellent reproducibility to deposit conjugated PEDOT-EM onto differentsurfaces, such as indium tin oxide coated polyethylene terephthalate(ITO-PET) substrate and gold coated silicon wafers (Au—Si). Thepolymerization process could be monitored and processed in a preciselycontrolled manner by simply adjusting the potential/current and reactiontime as, for example, described in Cui, X.; Martin, D. C. Sensor Actual.B Chem. 2003, 89, 92, the disclosure of which is incorporated herein.Three different methods, cyclic voltammetiy (CV), galvanostatic (GS),potentiostatic (PS) were tested and compared to polymerize EDOT-EM froman acetonitrile solution containing 100 mM monomer and 100 mM LiClO₄ asan electrolyte. The surfaces prepared from GS method showedsignificantly better homogeneity than those generated from CV and PSmethods. As illustrated in FIG. 2, during the GS electro-polymerizationprocess, the working potential decreased smoothly with the increase ofreaction time, indicating the decrease of overall impedance and theexcellent electrical conductivity of the deposited PEDOT-EM films. Froma mixed solution of acetonitrile and water (1/1 v/v) containing 60 mMmonomer (purity >90%) and 100 mM LiClO₄ as electrolyte, EDOT-EM waspolymerized with a PS method at 1.1 V. In general, high purity and highconcentration of monomer is preferred for successful polymerization.

Both PEDOT-EM and PEDOT control films were successfully polymerized onGold-coated silicon wafers. As shown in FIGS. 3A and 3B, electrochemicalimpedance spectroscopies (EIS) were recorded on both coated and uncoatedsubstrates to test the efficiency of the polymerization as well as thequality of deposited films. PEDOT-EM film showed excellent conductivityand low impedance (FIG. 3B), similar to that of PEDOT (FIG. 3A). Theimpedance of the PEDOT-EM coated substrate was over one order ofmaunitude lower than that of the uncoated gold at a broad range of lowfrequencies (FIG. 3B), indicating that the interfacial impedance of thecoated electrode was significantly decreased after PEDOT-EM coating,which is highly desired for many applications. In a previous report,others were unable to achieve efficient polymerization with EDOT-EM.Successful electrochemical polymerizationldeposition of EDOT-EM may, forexample, be the result of high polymerization rate and high molecularweight, along with many other factors, including the purity of monomer,concentration of monomer and electrolyte, type of solvent andelectrolyte, method of polymerizations, etc. After screening differentconditions, it is believed that the conditions used herein allowpolymerization with higher molecular weight so that the reaction is moreefficient to produce stable polymer films with high conductivity thatare comparable to PEDOT.

As described above, EDOT-EM may be functionalized with either acidcatalyzed hydro-alkoxy addition or thiol-ene click chemistry. It wasalso demonstrated herein that one could first polymerize the EDOT-EMinto PEDOT-EM and then post-functionalize the polymer on demand, with,for example, both small molecules and large proteins. In general, anymolecules in the “tool-box” equipped with free thiol (SH) units could bedirectly attached to the polymer through the click chemistry. After themonomer has been successfully electropolymerized into conductive polymerfilms, the film was first post-functionalized with a small molecule,3-mercaptopropionic acid (MPA). Attenuated total reflectance-Fouriertransform infrared (AIR FTIR) spectroscopy was taken before and afterfunctionalization of the films. PEDOT film was also tested and used as areference. As shown in FIG. 4, The FTIR spectra of the PEDOT andPEDOT-EM slightly differ from each other. For PEDOT-EM, an addition peakat 1549 cm⁻¹ was attributed to EM group, which was not seen in thespectrum of PEDOT. After thiol-ene functionalization of PEDOT-EM withMPA, the appearance of the bands from carbonyl C═O stretching, between1600 and 1800 cm⁻¹, confirmed the successful post-functionalization.

For specific biosensors and bioelectrode applications, surfacemodification with functional proteins may be desirable. To demonstratepost-polymerization functionalization of PEDOT-EM with largebiomolecules, primary neuron attachment was studied on PEDOT-EM coatedsubstrates pretreated with laminin, a protein that is widely used topromote neuronal attachment and neurite outgrowth. In a number ofstudies, Laminin was applied on nitrocellulose (NC, a substrateroutinely used in neuronal culture) surface, PEDOT and PEDOT-EM coatedITO-PET substrates, followed by washing in PBS three times. As shown inFIG. 5A, significant neuron attachment and growth was found on laminincoated NC surface as expected. Since PEDOT has no functional group to beconjugated with proteins, neuron growth was barely seen (FIG. 5C).Significant neuronal attachment and neurite extension were observed onthe PEDOT-EM sample, suggesting that laminin was successfully bound tothe PEDOT-EM surface (FIG. 5B). Although the conjugation efficiency hasnot been optimized, these studies demonstrate a convenient andcost-effective strategy for surface post-functionalization of conductingpolymer with biomacromolecules.

In another study, PEDOT-EM polymer film coated samples were incubatedwith 100 uL of 0.04 mg/ml laminin PBS solution for 1.5 hr, and thenrinsed with PBS for 3 times. Primary neuron cell culture was used tovalidate successful post-functionalization of PEDOT-EM -with lamininprotein. In these studies, direct coupling of the thiol groups onlaminin is achieved via reaction with the EM on the polymer. As shown inFIG. 6, no neuron cell attachments were observed on un-functionalizedbare PEDOT-EM surface. In contrast, PEDOT-EM post-functionalized withlaminin showed high density neuron cell attachments as well assignificant neurite growth and extension, indicating successful covalentlinking of laminin protein to PEDOT-EM. In FIGS. 6, high density neuronattachments were observed via blue DAPI stained nuclei (upper leftphotomicrograph), and neurite outgrowth and extension were observed viagreen, beta tubulin staining(lower left photomicrograph).

Another way to functionalize protein is to use crosslinkers that willreact to the EM on one end and amine group of the protein on the otherend. This methodology may, for example, be necessary when the thiolgroups on the protein are critical for its function. Another advantageof this approach is that the thiol-EM reaction may sometimes requirerelative harsh conditions, which may result in denaturing of theprotein. The use of crosslinkers may enable the use of relatively mildreaction conditions. In a number of studies, PEDOT-EM coated substrateswere immersed in 1,3-propanedithiol andN-γ-maleimidobutyryl-oxysuccinimide ester (GMBS) 10 mM ethanol solutionfor 1 hr sequentially. Afterward L1 or L1CAM protein (a transmembraneprotein) solution was incubated on the sample for another hour. Allsamples were rinsed with ethanol three times before use. FIG. 7demonstrates the success of the L1 immobilization on PEDOT-EM. Highdensity neuron attachments were observed via blue DAPI stained nuclei(right photomicrograph), and neurite outgrowth and extension wereobserved via green, beta tubulin staining (left photomicrograph).

In a number of further studies, PEDOT-EM was post functionalized withDNA aptamer functionalized with methylene blue. In several such studies,PEDOT-EM coated substrates were immersed in 0.25 mg/ml aptamer PBSsolution and kept at room temperature overnight. Before electro-chemicaldetection, all samples were rinsed with PBS for 3 times. Square wavevoltammetry was used to detect successful post-functionalization ofPEDOT-EM with DNA aptamer based on methylene blue oxidation. As shown inFIG. 8, before PEDOT-EM was functionalized, no methylene blue peak wasobserved around −0.3 V. After post-functionalization, a sharp and clearpeak was observed around −0.3 V (the oxidation potential of methyleneblue on the DNA aptamer), indicating the successful covalent linking ofaptamer to PEDOT-EM. PEDOT-EM may, for example, be functionalized with abiomolecule such as an aptamer to develop electrochemical biosensors.Aptamers are oligonucleotide or peptide molecules that bind to aspecific target molecule. The target binding to aptamer causes aptamerto change conformation which leads methylene blue to be closer orfurther away from the electrode surface. As a result, the change ofmethylene blue oxidation current can be related to target concentration.

Polymers hereof may thus be formed by polymerizing at least one monomerselected from the group of

via a polymerization reaction as described herein. The monomer may, forexample, be polymerized via electropolymerization as described above. Ifmonomer F (EDOT-EM) is polymerized, the resultant polymer may befunctionalized post-polymerization. Functionalization of the polymerafter synthesis may, for example, occur by reacting a compound with anexomethylene group of the polymer.

In the case that compound G is polymerized, compound G may be formed byreacting compound F with a functionalizing compound including a groupreactive with an exomethylene group of compound B, wherein the group Ris the residue of the reaction of compound F with the functionalizingcompound. In a number of embodiments, the monomer has a purity of atleast 90% or at least 95% in the polymerization reaction. In a number ofembodiments, the concentration of the monomer in the polymerizationreaction is at least 20 mM, 30 mM, 40 mM, 50 mM or 60 mM. In a number ofembodiments, electrolyte concentration is in the range of 10 mM to 1 M,or in the range of 50 mM to 200 mM. In a number of studies hereof, anelectrolyte concentration in the polymerization was at least 100 mM.Electrolytes suitable for use in the polymerization reactions hereofinclude, but are not limited to lithium, sodium, potassium,tetrabutylammonium or tetrabutylphosphonium salts with a variety ofcounter ions, including, for example, perchlorate, nitrate, acetate,chloride, bromide, iodide, hexafluorophosphate, tetrafluoroborate,tetraphenylborate, or polystyrene sultanate (PSS). Other suitableelectrolytes include ionic liquids such as ammonium-, pyridiniurn-,imidazolium-, or phosphonium-containing ionic liquids. In a number ofstudies, the electrolyte was, for example, LiClO₄.

Solvent systems/solvent mixtures for use in the polymerization reactionhereof include, for example mixtures of water and acetonitrile, ethanol,dimethylformamide, propylene carbonate, etc. Binary solvents (that is,mixtures of organic solvent and water) may, for example, include 5% to95% of water. In a number of studies hereof, a solvent system/solventmixture of acetonitrile and water was used. The acetonitrile and waterwere, for example, present in a 1:1 mixture.

A potentiostatic polymerization method was used in a number ofembodiments. A potential for such a reaction may, for example, be in therange of 0.6 V to 1.5 V, or in the range of 0.9 V to 1.2 V. In a numberof potentiostatic polymerizations hereof, the potential wasapproximately 1.1V. Electropolymerization methods such as potentiostaticor cyclic voltammetry (for example, at the same potential range as setforth above) may be used to synthesize the polymers hereof. Further,galvanostatic polymerization methods may be used with, for example, acurrent density in the range of 1 μA/cm² to 100 μA/cm². In a number ofembodiments hereof, galvanostatic polymerization may occur at a currentdensity of 50 μA/cm².

As described above, the exomethylene group of EDOT-EM or PEDOT-EM may bereacted with a compound having a group reactive with an exomethylenegroup of the polymer. The reaction may, for example, occur in a solutionstate. In a number of embodiments, the group reactive with theexomethylene group is a thiol group. The at least one compound may, forexample, be small molecule or a polymer. Examples of readily availablesmall thiol compounds for reaction with the exomethylene group include,but are not limited, to 3-mercaptopropionic acid,3-chloro-1-propanethiol, 1-mercapto-2-propanol, 3-mercapto-1-propanol,3-amino-1-propanethiol hydrochloride, 4-mercapto-1-butano,6-mercaptohexanoic acid, 6-mercapto-1-hexanol, 6-mercapto-1-hexanol,6-amino-1-hexanethiol hydrochloride, 8-mercaptooctanoic acid,8-mercapto-1-octanol, 8-amino-1-octanethiol hydrochloride,9-mercapto-1-nonanol, 11-mercaptoundecanoic acid, 11-mercaptoundecanamide, 11-Azido-1-undecanethiol, 11-mercapto-1-undecanol,11-Amino-1-undecanethiol hydrochloride, 11-mercaptoundecylphosphonicacid, 11-mercaptoundecylphosphoric acid, 12-mercaptododecanoic acid,1-(11-mercaptoundecyl)imidazole,(11-mercaptoundecyl)-N,N,N-ftimethylammonium bromide,11-(1H-pyrrol-1-yl)undecane-1-thiol, 6-(ferrocenyl)hexanethiol,12-mercaptododecanoic acid NHS ester, 16-mercaptohexadecanoic acid,16-mercaptohexadecanamide, 16-amino-1-hexadecanethiol hydrochloride,11-mercaptoundecylhydroquinone, triethylene glycolmono-11-mercaptoundecylether, (11-mercaptoundecyl)tetra(ethyleneglycol), 11-(fenocenypundecanethiol, (11-mercaptoundecyl)hexa(ethyleneglycol). The exomethyl group(s) may alternatively, for example, bereacted with zwitterionic amino acid cysteine for non-specificbiofouling resistance of the surface. Likewise, the monomers or polymershereof may be reacted with various biomolecules (for example, proteins,nucleic acids etc). The monomers or polymers may, for example, bereacted with a biomolecule such as an aptamer for use, for example, inreal time neurotransmitter detection, or with one or more peptides foruse, for example, in improving biocompatibility of the coated substratewith enhanced neuronal survival.

Once again, the exomethylene group of the monomer may be reacted withsuch functionalizing compound or other functionalizing compoundsreactive with the exomethylene group to create the substituent R setforth above in compound B. In forming functionalized EDOT polymers, theEDOT-EM monomer may be functionalized (as described above) prior topolymerization. In addition to thiol-ene chemistry, other reactions mayoccur, as known in the chemical arts, with the exomethylene group eitherprior to polymerization or after polymerization in forming afunctionalized EDOT monomer or polymer.

The foregoing description and any accompanying or incorporated drawingsset forth a number of representative embodiments at the present time.Various modifications, additions and alternative designs will, ofcourse, become apparent to those skilled in the art in light of theforegoing teachings without departing from the scope hereof, which isindicated by the following claims rather than by the foregoingdescription. All changes and variations that fall within the meaning andrange of equivalency of the claims are to be embraced within theirscope.

What is claimed is:
 1. A method of forming the compound:

comprising, the reaction:

in the presence of a base comprising teat-butyl lithium, lithiumdiisopropylamide, sodium hydroxide, potassium hydroxide, lithiumhydroxide, a potassium alkoxide or a sodium alkoxide to achieve a yieldof at least 90%, wherein X is a halo atom selected from the groupconsisting of Cl, Br and I. The method of claim 1 wherein a conjugateacid of the base has a pKa of at least
 17. 3. The method of claim 1wherein the reaction takes place under conditions to limit nucleophilicaddition reactions by the base.
 4. The method of claim 1 wherein thebase is a non-nucleophilic base.
 5. The method of claim 1 wherein thebase comprises potassium iert-butoxide and sodium tert-butoxide.
 6. Themethod of claim 1 wherein the base comprises potassium tert-butoxide. 7.The method of claim 6 wherein the reaction take place at roomtemperature.
 8. The method of claim 1 wherein the reaction occurs at atemperature in the range of approximately 0° C. to 100° C.
 9. The methodof claim 1 wherein the reaction proceeds to quantitative conversion. 10.The method of claim 1 wherein the base comprises sodium hydroxide andpotassium hydroxide.
 11. The method of claim 1 wherein the reaction hasa yield of at least 95%.
 12. The method of claim 1 wherein X is Cl. 13.A method of forming a polymer, comprising; polymerizing monomerscomprising at least one of exomethylene functionalized3,4-ethylenedioxythiophene or the reaction product of exomethylenefunctionalized 3,4-ethylenedioxythiophene and a compound including agroup reactive with the exomethylene group of exomethylenefunctionalized 3,4-ethylenedioxythiophene via a polymerization reaction,wherein the monomers have a purity of at least 90% in the polymerizationreaction.
 14. The method of claim 13 wherein the monomers have a purityof at least 95%.
 15. The method of claim 13 wherein the polymerizationreaction is an electropolymerization reaction and the method furthercomprises functionalization of the polymer after synthesis by reacting acompound with an exomethylene group.
 16. The method of claim 13 whereinthe monomers have a concentration of at least 20 mM in thepolymerization reaction.
 17. The method of claim 13 wherein the groupreactive with the exomethylene group is a thiol group.
 18. The method ofclaim 13 wherein the compound including the group reactive with theexomethylene group is selected from the group consisting of3-mercaptopropionic acid, 3-Chloro-1-propanethiol,1-mercapto-2-propanol, 3-mereapto-1-propanol, 3-amino-1-propanethiolhydrochloride, 4-mercapto-1-butano, 6-mercaptohexanoic acid,6-mercapto-1-hexanol, 6-mercapto-1-hexanol, 6-amino-1-hexanethiolhydrochloride, 8-mercaptooctanoic acid, 8-mercapto-1-octanol,8-amino-1-octanethiol hydrochloride, 9-mercapto-1-nonanol,11-mercaptoundecanoic acid, 11-mercaptoundec anamide,11-azido-1-undecanethiol, 11-mercapto-1-undecanol,11-amino-1-undecanethiol hydrochloride, 11-mercaptoundecylphosphonicacid, 11-mercaptoundecylphosphoric acid, 12-mercaptododecanoic acid,1-(11-mercaptoundecyl)imidazole, (11-mercaptoundecyl)-N,N,N-trimethylammonium bromide,11-(1H-pyrrol-1-yl)undecane-1-thiol, 6-(ferrocenyl)hexanethiol,12-mercaptododecanoic acid NHS ester, 16-mercaptohexadecanoic acid,16-mercaptohexadecanamide, 16-amino-1-hexadecanethiol hydrochloride,11-mercaptoundecylhydroquinone, triethylene glycolmono-11-mercaptoundecyl ether, (11-mercaptoundecyl)tetra(ethyleneglycol), 11-(ferrocenyl)undecanethiol, (11-mercaptoundecyl)hexa(ethyleneglycol), or a biomolecule.
 19. The method of claim 18 wherein thebiomolecule is a zwitterionic amino acid cysteine, a peptide, a protein,or a nucleic acid.
 20. A method of forming a polymer, comprising:polymerizing monomers comprising at least one of exomethylenefunctionalized 3,4-ethylenedioxythiophene or the reaction product ofexomethylene functionalized 3,4-ethylenedioxythiophene and a compoundincluding a group reactive with the exomethylene group of exomethylenefunctionalized 3,4-ethylenedioxythiophene via a polymerization reaction,wherein the monomers have a concentration of at least 20 mM in thepolymerization reaction.